This DNA Vaccine core proposal is designed to further research that has resulted from a collaboration between Apollon and the Institute for Biotechnology and Advanced Medicine at the University of Pennsylvania. These efforts recently yielded reports describing the successful immunization of several species with vectors that express HIV-1 derived proteins. Mice, rabbits, cynomolgus macaques and chimpanzees were inoculated with a vector that encodes the HIV-1MN gp160 and Rev proteins and/or a vector that expresses the Gag and Pol proteins of HIV-1HXBII. Each vector, induced both cellular and humoral immune responses specific o the expressed antigens in the injected animals. Furthermore, this technology has resulted in the protection of the immunized cynomolgus macaques from early viral replication following high dose challenge with HIV-1SF2. Most recently, both vectors have been tested for safety in a series of Phase I/II clinical trials conducted in HIV-1-infected patients and, also, in normal volunteers. The aim of the research described here is to optimize the activity of the facilitated DNA-based vaccines that have been developed and tested to date. It is possible that co-infection of vectors that express either a cytokine or a co-stimulatory molecule along with the vaccine vectors encoding the HIV-1-derived antigens could result in a stronger immune response providing a more effective basis for the development of a prophylactic vaccine for HIV-1 infection. For the present proposal, clinical vaccine vectors that express the subunits of IL-12, the B7.2 molecule or other co-stimulatory molecules will be constructed. These vectors will be made using the Apollon backbone(s) that have already been used in clinical trials and that were designed to be safe upon injection in vaccines or patients. The immediate objectives of this Molecular Biology Core proposal will be (i) construction and structural analysis of the plasmids; (ii) evaluation of protein expression in human rhabdomyosarcoma and COS-7 cells that have been transfected with the construct of interest; (iii) the production of large scale amounts of the product plasmids according to GLPs and cGMPs for animal and clinical studies; (iv) evaluation of the quality and preclinical safety of the clinical vectors prior to clinical. The long- term goal of this work is the development and commercialization of GENEVAX-HIV for the possible prophylaxis of HIV-1 infection.